4. Discussion
As a potent mitogenic factor, FGF2 has been extensively explored for potential application in wound healing (Li et al. , 2016). However, FGF2 can be rapidly degraded by enzymes and rapid renal filtration in vivo, thereby limiting its clinical value. Site-specific protein PEGylation can minimize reduction in biologic activity while improving pharmacokinetic properties.
In this study, we found that the mutation of either cysteine on the surface of FGF2 is not detrimental to protein activity, which is consistent with the results of previous study (Kang et al. , 2010). However, the mutation of surface sites near the heparin and receptor binding domains slightly reduced protein activity. The results indicate that surface-exposed sites that are spatially separated from both binding domains may be better candidates for modification. The three conjugates obtained by PEGylation showed different bioactivities. In particular, compound 6 , PEGylated at a site spatially separated from both the heparin and receptor binding regions, exhibited the strongest activity. Compound 5 with the PEGylated site close to the receptor binding domain showed the second-highest activity, and compound 7 with the site of PEGylation near the heparin binding domain was the weakest. The heparin binding region may be more conserved, and steric hindrance due to the presence of PEG polymers may affect heparin binding. Heparin makes numerous contacts with both FGF2 and FGFR, thus augmenting FGF2-FGFR binding (Sarabipour et al. , 2016), and it also interacts with FGFR in the 1:1 FGF2-FGFR complex to facilitate FGF2-FGFR dimerization, with increased intracellular receptor tyrosine kinase activity that triggers various downstream signaling pathways (Schlessinger et al. , 2000). In addition to these roles in both FGFR binding and dimerization, heparin may function directly as a receptor for FGF2 (Chua et al. , 2004).
In summary, based on the structure of FGF2, four surface-exposed sites close to or far from the receptor/heparin binding regions, were selected and mutated to cysteine or alanine, allowing assay of PEGylation of specific residues. All PEGylated FGF2 conjugates exhibited significantly improved stability. Among these conjugates, compound 6 , modified far from the heparin and receptor binding domains, exhibited the strongest biological activity. This conjugate effectively promoted the proliferation and migration of epithelium and dermal cells, and enhanced the activity of vascular regeneration to accelerate wound healing. These results together suggest that compound 6 may be a promising candidate for the treatment of cutaneous wounds. Overall, structure-guided site-specific PEGylation offers a good strategy to develop improved proteins for use as therapeutics.